The present invention is directed to an improved method of crystallizing human serum albumin (“hSA”)(herein, hSA will be used interchangeably with the term human serum albumin). This process is preferably done to enhance purification procedures for recombinant hSA that can then be utilized in therapeutic applications or as an excipient in pharmaceutical preparations. With regard to pharmaceutical preparations human albumin as purified herein can be used as a therapeutic agent or as an excipient. In either case suitable formulations can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990)), and in INTRODUCTION TO PHARMACEUTICAL DOSAGE FORMS (4th Edition, Lea & Febiger, Philadelphia (1985)), each of which is incorporated herein by reference.
For therapeutic applications of hSA the objective of albumin administration is primarily to maintain circulating plasma volume by maintaining the plasma colloid oncotic pressure, and to treat otherwise resistant severe edema by making intracavital and interstitial fluids move into the blood vessels.
Albumin products are used to achieve transient improvement of the condition by replenishing albumin in pathological conditions attributable to acute hypoproteinemia, and pathological conditions resulting from chronic hypoproteinemia which is resistant to other methods of treatment.
Albumin was the first natural colloid composition for clinical use as a blood volume expander, and it is the standard colloidal agent for comparison with other colloid products. Some of the specific medical indications in which albumin may be used to increase intravascular oncotic pressure and thereby expand intravascular volume in patients include: hypovolemic shock; severe burn injury; adult respiratory distress syndrome (ARDS); ascites; liver failure; pancreatitis and in patients undergoing cardiopulmonary bypass. (Cochrane et al., 1998). Albumin may also be used to treat neonatal hyperbilirubinemia, hypoproteinemia, and nephrotic syndrome. (Vermeulen et al., 1995).
The albumin portion of human blood serves three primary physiologic roles: (1) maintenance of plasma colloid osmotic pressure, (2) transport and sequestration of bilirubin, and (3) transport of fatty acids and other intermediate metabolites such as hormones and enzymes. (Peters, T et. al.,). Because albumin accounts for approximately 80% of the oncotic pressure of plasma, a 50% reduction in serum albumin concentration consequently produces a 66% decrease in colloid oncotic pressure. (Rainey T. G., et al., 1994). In critically ill patients, risk of death is inversely related to serum albumin concentration. (Cochrane et al., 1998). Goldwaser and Feldman estimate that for each 2.5 g/dL decrease in serum albumin concentration, there is a 24%–56% increase in the risk of death. (Goldwaser et al., 1997). This estimate was made after adjusting for other co-variants (e.g., renal function, serum trans-aminase, lactic acidosis), and it strongly indicates that albumin infusion may have a direct cytoprotective effect. (Cochrane et al., 1998).
Given the above, it is clear that hSA is perhaps the best known of all the plasma proteins judging both by the amount of scientific literature available describing it as well as through the number of industrial uses it enjoys. However, this abundant amount of knowledge is focused primarily on its physiology and the clinical use of albumin, not the methodology used to purify it or sourcing the molecule from anything other than plasma fractionation. The best-known and still widely used purification methods were developed by Cohn and co-workers 60 years ago (Cohn E. J. et al., 1947). The Cohn plasma fractionation method is primarily used to produce purified plasma products for a wide variety of clinical uses. Cohn also developed a widely used crystallization process which utilizes principles similar to those well-known from plasma fractionation processes for use with human serum albumin. However, the process has significant inefficiencies and often does not provide an adequate supply of highly purified hSA.
Effect of pH
The effect of pH is one of the major factors in protein crystallization. Usually protein crystals have a well-defined minimum point of solubility at a specific pH. In the general literature of protein crystallization, it is most often the case that this minimum solubility is at the isoelectric point of the target protein. However, hSA is highly soluble at its isoelectric point, across a wide range of ionic strengths. Thus, the crystallization properties of albumin are much more complex than those of many other proteins making reliable crystallization and/or purification problematic.
Albumin has a varying isoelectric point depending on the chemical treatment that it has received. With a full complement of six bound fatty acids hSA's pI is normally 4.6, however, when fully de-fatted its pI may be as high as 5.6. Therefore, the crystallization properties of hSA vary as between its “native” and de-fatted states and the reported optimum pH for the crystallization of hSA itself varies substantially in the literature from a low of pH 4.6 to a high of pH 8.0 and may be highly dependent upon the molecular state of hSA in a batch-by-batch basis. Thus, the wide range of pH that is considered to be optimal for the crystallization of hSA present in the literature is confusing and apparently relies on various precipitating reagents, each of which is utilized having a variable concentration and which may be optimal for only one of the possible molecular states of hSA.
For example, with hSA at low ionic strength, like that expected in the Cohn alcohol process, crystallization proceeds optimally at pH 4.9–5.3 which is close to the isoelectric point of native albumin. In conditions with a higher ionic strength and when strongly buffered the optimal pH for crystallization by PEG solutions is 7.4. In sum, the reported pH effects for optimal crystallization of hSA are dependent on the reagent composition in such a seemingly irregular way that solid conclusions cannot be made by reference to the prior art and prior art methodology. In fact, given the status of the teachings of the prior art, every new reagent and technique must be laboriously optimized according to a specific pH or other single variable to be kept constant while crystallization conditions are worked out.
Effect of Precipitating Reagents
It should be noted that hSA has a very high solubility in varying salt concentrations. It can be precipitated or crystallized at low ionic strength with added ethanol (Cohn process; Cohn E. J. et al., 1947) or other solvents. Alternatively, salting out with very high salt concentration is possible, and the early literature mostly used ammonium sulfate or ethanol is described. In the more recent literature PEG solutions of various molecular weights have be utilized widely. However, the reagents present in the literature are unacceptable for the clinical use of the resulting hSA because of the remaining contaminants. Of the prior art precipitating reagents only ethanol and ammonium sulfate are useful in the production of has. However, they both have significant practical problems. Crystallization with ethanol requires the addition of toxic organic modifiers such as benzene or heavy metals. Ammonium sulfate is not a suitable salt for final albumin formulation, and would thus need to be removed.
Effect of Specific Reagents
In addition to its other characteristics albumin has an extraordinary capacity to combine and attach to a wide variety of smaller molecules and ions. The association of various long chain alcohols and fatty acids with hSA strongly affect the crystallization profile of molecular hSA and again act to make the production of clinical grade human serum albumin highly variable and unpredictable. Examples of reagents capable of significantly effecting the crystallization profile of hSA include: decanol, palmitic acid and caprylic acid.
Effect of Temperature
In should also be noted that prior art attempts to crystallize hSA in ethanol solutions have typically been made at low temperatures in the range of 0–10° C. High salt and PEG procedures are often made at a wider temperature range of 4–20° C. In these prior art efforts it is not clear what the effect of temperature really is on albumin precipitation. In ethanol the crystal solubility is seemingly lower at low temperature. The effect of temperature is not clearly described in the PEG and salt methods found in the prior art. For production efficiency and commercial viable processes temperature is one of the major factors. Overall, the significance of temperature is not explained or disclosed by the prior art.
Kinetics and Seeding
According to the prior art, the time period needed for the crystallization of albumin in a given reaction to be complete can take up to several days. However, of the prior art methods those employing ethanol may be the most rapid, requiring only 12 to 24 hours to initiate crystallization. Also according to prior art methods the actual crystallization of albumin may not be possible at all without additives including seeding a reaction mixture with crystals formed from a prior reaction. In addition, the methods of crystallization relying on PEG, may or may not utilize this type of additive. It should be noted that seeding does speed up the crystal growth significantly, though given the confusion in the art generally there are no references that can be utilized which give consistently reliable results or generate a high yield of crystal.
TABLE 1Example List of Crystallizing Reagents and Conditions Found in the Literature.bufferstemp.PrecipitantsAdditives0.05–0.1 MpH° C.References(NH4)2SO4 50%Decanolphosphate4.6–7.74–6Haupt, H. and Heide, K. Klin.Na2SO4 15–20%acetate5.0–6.820Wochenschr. (1967), 45(14),K-phosphate 2.2 M5.94–6pp. 726–729.Na-phosphate 3 M6.8PEG 180–800K-phosphate4.6–7.24(1) Carter, D. C. EP 0 35740%Na-acetate6.8857 A1 andPEG 400Na-citrate, Tris5.5–7.2(2) Carter, D. C. et. al.40%Science 244 (4909) (1989)p. 1195PEG 3350long chainphosphates7.522(1) Carter, D. C.17.5%fatty acidsNa-acetate4.6–8.0U.S. 5.585.466 andNa-citrate, Tris7.0–7.5(2) Carter, D. C. et. al. Eur. J.Biochem. 226(3)(1994)p. 1049PEG 3350K-phosphate74Bhattacharya et al J. Biol.28–30%Chem. 275(49)(2000) p.38731PEG 400, 4000K-phosphate5.0–5.515–20Sugio, S. et. al. Prot. Eng.20–38%7.0–8.012(6) (1999) p. 439(NH4)2SO4DecanolK-phosphate64Rao, S. et. al. J. Biol. Chem.45% saturated251(10) (1976) p. 3191MPDDecanol5.21McClure, R. J. et. al. J. Mol.0.1%Biol. 83(4) (1974) p. 551Ethanol5.22Low, B. W. J. Am. Chem.Soc. 74(1952) p. 4830(NH4)2SO4Decanol6.81Low, B. W. and Weichel,54% saturated.0.2%E. J. J. Am. Chem. Soc.73(1951) p. 3911MethanolNumerousacetate4.4–6.5  −5 ---Lewin, J. J. A M. Chem. Soc.Ethanol 5–30%compounds4.9–5.1+573 (1951) p. 3906Acetoneheavy metalsEthanol, moleCHC134.9  −5 ---Cohn, E. J. et. al. J. Am.fractions 0.02–Decanol,5.3+10Chem. Soc. 69 (1947) p. 17530.163benzene,Ethanol 15%Decanol5.2<0Hughes, W. L. J. Am. Chem.HgC13Soc. 69 (1947) p. 1836Prior Art Methodology with Mineral Salts
The prior art (Haupt and Heide (1967)) provided methods to crystallize human serum albumin with various mineral salts including: 50% saturated (NH4)2SO4; 15–20% Na 2SO4; 2.2M K-phosphate pH 6.8 and 3M Na-phosphate pH 5.0. Decanol was found to be a necessary crystallization aid in these prior art methods. Other fatty alcohols with more than five carbon atoms in their molecular backbone were found to be useful also. However, the crystallization conditions and procedures were very sparingly described. No material balances were presented. On the basis of the data available from this citation it is not possible to perform crystallization of albumin in a sufficiently controlled or reliable way.
A review of the prior art literature indicates that while there are several methods of crystallization proposed the relevant citations do not teach a process that is efficient at an industrial or commercial scale, teach a method that is unavailable for use in the production of a therapeutic product or excipient, or provide a process only useful in the production of single crystals useful only in x-ray diffraction studies. Thus, the limitations of the prior art prevent the development of the extensive knowledge of crystallizing conditions necessary in the design of a large-scale crystallization processes for hSA in a therapeutic, pharmaceutical excipient, or medical adjuvant role. Moreover, the prior art does not provide teachings that provide for the use from sources other than human plasma. Therefore, a need exists to understand the physical and chemical conditions which produce crystals of albumin reliably and on an commercial scale from a variety of sources.